In healthy individuals, central and peripheral immune tolerance mechanisms prevent autoimmune disease. One or more of these mechanisms is disrupted in Type 1 diabetes (T1D) patients, resulting in autoimmune destruction of insulin-producing pancreatic beta cells. In the NOD mouse model, defects in thymic negative selection have been strongly implicated in disease development. Introduction of an insulin-reactive T-cell receptor (TCR) derived from a T1D patient to peripheral T cells of humanized mice can initiate diabetes, consistent with the notion that escape of autoreactive T-cells from negative selection can promote disease. On the other hand, beta cell antigen-reactive T-cells have also been isolated from the blood of healthy control (HC) individuals, raising the possibility that defective peripheral tolerance mechanisms may be most important in promoting T1D. Genetic studies have identified over 60 risk variants for T1D, including associations with genes implicated in deletion of developing autoreactive T cells during thymic negative selection. To date, however, there has been no method available to model human patient-specific thymic selection, resulting in uncertainty about the role of defects in this pathway in promoting human autoimmune diseases. We hypothesize that there are specific hematopoietic stem cell (HSC)-intrinsic and thymus-intrinsic genetic variants in T1D individuals that lead to the failure of negative selection of diabetogenic T-cell receptors (TCRs) in the thymus. We have established a Personalized Immune (PI) mouse model that allows us to generate an adult human's immune system de novo in immunodeficient NOD/LtSz-scid IL2R gamma null mice (NSG) mice receiving patient HSCs and a partially HLA-matched fetal thymus graft. We have also demonstrated that adult patient HSCs can be lentivirally transduced to express a TCR and subsequently used to reconstitute NSG mice or human thymus tissue in thymic organ culture assays. We have demonstrated that a diabetogenic insulin B(9-23)/HLA-DQ8- specific autoreactive TCR is normally deleted within the thymus of humanized mice constructed with HLA-DQ8+ thymic tissue and HSCs. With these data and with access to HSCs from T1D and HC donors and to thymi from the network for Pancreatic Organ Donors with Diabetes (nPOD), we have demonstrated the feasibility of our proposed study. We will use these innovative in vitro and in vivo models to assess whether or not there is a defect in thymic negative selection in immune systems derived from T1D patients and determine whether such defects are intrinsic to the patient HSCs or to T1D thymic tissue. We will associate such defects with genetic risk variants and address hypotheses about the impact of specific variants that may impact negative selection in an HSC- or thymic tissue-dependent manner. These studies will not only provide unprecedented information on the role of defective negative selection in T1D pathogenesis and the genetic determinants of this defect, but will also establish a patient-specific model for assessing negative selection and determining its role in the development of multiple autoimmune diseases.